1. Field of the Invention
This invention concerns novel agents acting as co-factors for replacement of NAD(P)+/NAD(P)H co-enzyme systems in enzymatic oxido-reductive reactions. In particular, the invention concerns water soluble or partially water soluble agents mimicking the action of NAD(P)+/NAD(P)H system in enzymatic oxidation/reduction of substrates into reduced or oxidized products. The invention further concerns a method for selection and preparation of the mimicking agents for replacement of NAD(P)+/NAD(P)H system. Finally, the invention concerns a device comprising co-factors for replacement of NAD(P)+/NAD(P)H system.
2. Background and Related Disclosures
Due to their high selectivity and low environmental impact, enzymatic reactions are critically important. More than 35% of all known enzymes, however, need coenzymes or co-factors to enable their proper function. In particular, oxido-reductases, enzymes which catalyze oxidation and/or reduction of a substrate to a product, require the regeneration of NAD(P)+ and NAD(P)H co-factors for energy delivery. However, the utility of NAD(P)+/NAD(P)H oxido-reduction system for industrial purposes is impractical because the endogenous supply of NAD+/NADH co-factors is limited, and recovery of these co-factors from the natural sources or their synthetic preparation is expensive. Moreover, the energy delivery to the NAD(P)+/NAD(P)H regeneration processes requires redox catalysts and, therefore, the regiospecific regeneration of NAD(P)H at high rates is necessary. Such co-factor regeneration has been the limiting step for the economic utilization of NAD(P)+/1,4-NAD(P)H system in enzymatic chiral synthesis reactions, particularly for larger volume and more energy intensive processes.
Development of practical methods for the regeneration of the co-enzyme 1,4-NADH, the reduced form of nicotinamide adenine dinucleotide (NAD+), has continued to be of significant importance in the biocatalysts field, where enzymatic reduction reactions are used for chiral organic compound synthesis (Appl. Biochem. Biotech., 14:147 (1984) and J. Chem. Soc. Perkin Trans., 1:967 (1995)).
Conversion of NAD+ to 1,4-NADH by enzymatic, chemical, photochemical, or electrochemical methods has been extensively studied.
In order to develop faster rates and a more economical regeneration process, various transition metal hydrides have been studied as catalysts for the regioselective reduction of NAD+ and NAD+ models to the 1,4-NADH derivatives as described in Organometallics, 10:1568 (1991), J. Am. Chem. Soc., 116:2141 (1994) and Nat. Struct. Biol., 3:213 (1996)).
In the most illustrative example, Angew. Chem. Int. Ed. Engl., 29:388 (1990), describes the use of in situ generated [Cp*Rh(bpy)(H)]+(bpy=2,2xe2x80x2-bipyridyl), for the regiospecific reduction of NAD+ to 1,4-NADH, and then demonstrated the co-factor regeneration process with enzymatic, chiral reduction reactions.
While the above mentioned reduction of NAD+ by [Cp*Rh(bpy)H]+ was shown, at that time, to be highly regiospecific for 1,4-NADH, the mechanistic details of this important co-factor conversion were not known (Chem. Ber., 122:1869 (1989) and Orcanometallics (1991), supra). Some insights were only recently provided by inventors in a preliminary communication published in Angew. Chem. Int. Ed., 38:1429 (1999).
The above described methods were used solely for regeneration of the overly expensive NAD+/NADH system which is not suitable for larger volume and more energy intensive oxido-reductive enzymatic processes. For these purposes, the industrial utilization of NAD(P)+/NAD(P)H system is limited.
Therefore, it would be highly advantageous to have available new methods and/or agents which would replace expensive NAD(P)+/NAD(P)H system. Such methods and/or agents for complete replacement for NAD(P)+/NAD(P)H would greatly increase the capacity of enzymatic oxido-reduction reactions to deal with large volumes of products.
It is, therefore, a primary objective of the current invention to provide effective and inexpensive agents acting as co-factors for replacement of the NAD+/NADH system in oxidation/reduction enzymatic reactions and a method of use thereof.
All patents, patent applications and publications cited herein are hereby incorporated by reference.
One aspect of the current invention is a method for effective replacement of NAD(P)+/NAD(P)H system in enzymatic oxido-reductive reactions with novel agents of the invention.
Another aspect of the current invention is a method for replacement of NAD(P)+/NAD(P)H system in enzymatic oxido-reduction reactions comprising replacing the NAD(P)+/NAD(P)H with novel water soluble or partially water soluble agents acting as co-factors for oxido-reduction reactions and mimicking the action of NAD(P)+/NAD(P)H system.
Still yet another aspect of the current invention concerns a group of novel agents acting as co-factors for replacement and closely mimicking the action of NAD(P)+/NAD(P)H system in oxido-reduction enzymatic reactions, said agents depicted by the formula 
wherein R is xe2x80x94CN, xe2x80x94C(O)NH2, xe2x80x94C(O)NHCH3, xe2x80x94C(O)N(C2H5), xe2x80x94C(S)NH2, xe2x80x94C(O)CH3, or xe2x80x94C(O)OCH3;
wherein R1 is xe2x80x94(CH2(CH2O)nYR2, ribose-Yxe2x80x94R2, or 
wherein Y is xe2x80x94OPOOxe2x80x94, xe2x80x94OBO2xe2x80x94, xe2x80x94OSO2xe2x80x94, CH3NHxe2x80x94, xe2x80x94(CH2)nNHxe2x80x94, adenine, or imidazole;
wherein R2 is H, xe2x80x94CH3, xe2x80x94(OCH2CH2)n, xe2x80x94(NCH2CH2)nxe2x80x94 or xe2x80x94[(Nxe2x95x90P(OCH3)2]n;
wherein X is xe2x80x94OCH3, xe2x80x94CF3, xe2x80x94O(CH2CH2O)n or xe2x80x94OPOOR2;
wherein R3 is H, xe2x80x94CH3, xe2x80x94(OCH2CH2)n, xe2x80x94(NCH2CH2)nxe2x80x94 or xe2x80x94[Nxe2x95x90P(OCH3)2]n; and
wherein n is 1-2000; or a salt thereof.
Yet another aspect of the current invention concerns novel water soluble, or partially water soluble biomimics suitable for replacement of NAD(P)+/NAD(P)H system in reductive enzymatic reactions represented by N-substituted nicotinamide compounds and structurally related compounds and their derivatives chemically reduced by a catalyst formed by a reaction of a rhodium compound [Cp*Rh(bipyridyl)H]+ or other metal comprising compound with a reducing agent, said compounds functioning as co-factors for replacement of NAD(P)+/NAD(P)H system.
Yet another aspect of the current invention concerns novel water soluble, or partially water soluble biomimics suitable for replacement of NAD(P)+/NAD(P)H system in reductive enzymatic reactions represented by xcex2-nicotinamide ribose-5xe2x80x2-methyl phosphate and structurally related compounds and their derivatives chemically reduced by a catalyst formed by a reaction of a rhodium compound xcx9cCp*Rh(bipyridyl)H]+ or other metal comprising compound with reducing agents, said compound functioning as co-factors for replacement of NAD(P)+/NAD(P)H system.
Yet another aspect of the current invention concerns novel water soluble, or partially water soluble biomimics suitable for replacement of NAD(P)+/NAD(P)H system in oxidative enzymatic reaction by N-substituted nicotinamide compounds that are oxidized by electrodes, photons, chemical oxidants or enzymes.
Yet another aspect of the current invention concerns novel water soluble, or partially water soluble biomimics suitable for replacement of NAD(P)+/NAD(P)H system in oxidative enzymatic reaction by xcex2-nicotinamide ribose-5xe2x80x2 methyl phosphate nicotinamide compounds that are oxidized by electrodes, photons, chemical oxidants or enzymes.
Still yet another aspect of the current invention is a device, such as a biosensor or a chemical membrane bioreactor, useful for replacement and/or regeneration of NAD(P)+/NAD(P)H system.